Genetically Detoxified Pertussis that Maintains Intrinsic Adjuvant Activity

ABSTRACT

The invention is directed to a method for the production of a  pertussis  vaccine and, in particular, a detoxified  pertussis  vaccine comprising detoxified  pertussis  toxin (PT), detoxified  pertussis  lipopolysaccharide (P-LPS), and detoxified  pertussis  adenylate cyclase toxin (P-ACT). The invention is also directed to the manufacture of a vaccine or the invention and methods for the administration of a detoxified vaccine to patients.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/024,577 of the same title and filed Jul. 15, 2014, the entirety ofwhich is hereby incorporated by reference.

BACKGROUND

1. Field of the Invention

The invention is directed to methods for the production and manufactureof vaccines for the treatment or prevention of infection due toBordetella pertussis and, in particular, the invention is directed to adetoxified pertussis vaccine and methods for the administration ofdetoxified vaccines to patients.

2. Description of the Background

The bacterium Bordetella pertussis is the most common causative agentfor the disease referred to as whooping cough. Whooping cough is arespiratory disease that can infect both adults and children. Althoughtreatable with antibiotics, it can be a serious disease for infantsespecially. Clinically the disease is characterized by paroxysms ofrapid coughs followed by inspiratory effort, often associated with acharacteristic ‘whooping’ sound—thus the name. In more serious cases,there can be pneumonia and complications caused by pneumonia, and alsobrain oxygen deprivation which can lead to brain damage and death.

Although most often due to B. pertussis, about 5-10% of cases ofwhooping cough may be caused by a serologically related microorganism(e.g., B. parapertussis) (see, Mertsola (1985) Eur J Clin Microbiol 4;123; Lautrop (1971) Lancet 1(7711) 1195-1198). Infections due to B.parapertussis are clinically mild as compared with B. pertussis and, dueto the cross reactivity with B. pertussis makes B. parapertussisdifficult to diagnose as the causative agent.

The first generation of vaccines against B. pertussis were whole cellvaccines (referred to as wP vaccines), composed of bacteria which weretreated with chemicals such as formaldehyde to kill the cells andinactivate the toxic materials. Highly efficacious, these vaccines wereintroduced in many countries in the 1950s and 1960s. Although successfulat reducing incidence of whooping cough, a major problem with wPvaccines was the high level of reactogenicity, which commonly includedthe side effect of fever and local reactions.

The need for a more defined vaccine was recognized which led to thedevelopment of a vaccine comprising lesser number of highly purifiedantigens from different microorganisms. The result was what wasgenerally referred to as the component vaccine and contains immunogenicportions of diphtheria, tetanus and pertussis (DTP). Initial DTPvaccines contained pertussis endotoxin, surface lipooligosaccharide(LOS) of B. Pertussis (DTwP vaccines). LOS is a low molecular weightform of bacterial lipopolysaccharides (LPS). Although effective, thesevaccines produced a number of deleterious side effects.

In the 1990's, newer pertussis vaccines were developed that, althoughless uniformly immunogenic, include only a few selected pertussisantigens, namely toxins and adhesins of B. pertussis. Although lessdefined as compared to component vaccines, these acellular vaccines(DTaP vaccines) are less likely to provoke side effects and have beenapproved by the FDA for administration to adults and children. Vaccineshave been developed recently, also for administration to adults andchildren, which combine the tetanus and diphtheria toxoids withacellular pertussis vaccine. These (Tdap) vaccines, although lessimmunogenic than wP vaccines, contain reduced amounts of pertussisantigens compared to DTaP vaccines and, thus, have fewer side effects.Acellular vaccines containing purified B. pertussis proteins were lessreactogenic and have been adopted for many vaccination programs aroundthe world. Acellular vaccines typically containing pertussis toxin (PT),filamentous haemagglutinin (FHA) and quite often pertactin (PRN), arewidely used and provide effective protection from the severity ofwhooping cough.

Despite vaccination, whooping cough remains an endemic disease (Mooi etal (2001) Emerging Infectious Diseases 7; 526), and has re-emerged inAustralia, Canada and various areas of Europe; countries with highlyvaccinated populations. A comparison of pre-vaccination strains withstrains isolated from these areas has shown antigenic drift,particularly in PT and PRN (Mooi et al (1998) Infection and Immunity 66;670). It has become accepted that current vaccinations protect againstsevere disease, but do not eliminate the B. pertussis infection (Cherryet al (1998) Vaccine 16; 1901, Hewlett and Halperin (1998) Vaccine 16;1899,

Storsaeter et al (1998) Vaccine 16; 1907). The defense mechanismsassociated with B. pertussis allow the organism to escape theimmunological processes that would otherwise destroy the microorganism.

Not surprisingly, vaccination using whole cell B. pertussis vaccines(Pw) have been shown to be protective against B. parapertussis infectionas well, most likely due to an immunological similarity of theorganisms. B. parapertussis infection in unvaccinated infants may leadto severe and fatal complications, whereas in individuals vaccinatedwith Pw, a milder, often subclinical course of whooping cough is seen(Long et al (1990) Pediatric Infect Dis J 9; 700). Thus, acellularpertussis vaccines containing only two or three purified proteins shouldreduce the ability of vaccination to protect against B. parapertussis.Further improved acellular vaccines against whooping cough are requiredthat combine low reactogenicity with an ability to elicit a protectiveresponse against Bordetella, particularly with regard to infections ofB. pertussis and B. parapertussis.

Thus, a need currently exists for an effective pertussis vaccine that isuniformly immunogenic and protective against infection that has few orno side effects.

SUMMARY OF THE INVENTION

In general, the invention is directed to vaccines and methods for makingand administering vaccines that are effective against pertussis.

One embodiment of the invention is directed to vaccines for thetreatment or prevention of an infection in a mammal comprising: adetoxified pertussis toxin protein; an enzyme treated LPS; and adetoxified adenylate cyclase protein. Preferably the vaccine is for thetreatment or prevention of the infection of B. pertussis. Preferably thedetoxified pertussis protein contains a mutation in one or more ofsubunits S1-S6 of B. pertussis, such as, for example, a substitution inS1 of glutamic acid for glycine at amino acid position 129 and asubstitution of arginine for lysine at amino acid position 9. Preferablyenzyme treated LPS comprises LPS treated with one or more alkalinephosphatases or one or more deacylases and the one or morephosphorylaese comprise Antarctic phosphatase or lambda proteinphosphatase. Also preferably, the detoxified adenylate cyclase proteincomprised a dipeptide inserted between amino acids 188 and 189 of nativeP-ACT. Preferably the vaccine further comprises a pharmaceuticallyacceptable carrier such as, for example, water, glycerol, alcohol,propylene glycol, fatty alcohols, triglycerides, fatty acid esters,mineral oils, liquid petrolatum, isopropylpalmitate, polyethyleneethanol, polyoxyethylene monolauriater, sodium lauryl sulfate, ananti-oxidant, a humectant, a viscosity stabilizer or modifier, acolorant or a flavoring agent. Preferably the vaccine produces no sideeffects, such as, for example, no fever or inflammation at the site ofadministration to the individual. Another embodiment of the invention isdirected to methods of treating or preventing an infection comprisingadministering a therapeutically effective amount of the vaccine of theinvention to a mammal. Preferably the infection is caused by one or moreof B. pertussis, B. parapertussis or a serotype or strain of B.pertussis or B. parapertussis, and the vaccine comprises a detoxifiedpertussis toxin protein; an enzyme treated LPS; and a detoxifiedadenylate cyclase protein. Preferably administering is via injection,intramuscular injection, intravenous injection, oral administration,nasal administration, or a combination thereof. Preferablyadministration may be as a liquid, a tablet, a pill or a spray.Preferably the therapeutically effective dose is an amount of vaccinewhich produces an immune response in the mammal that is protectiveagainst and/or treats the infection. The therapeutically effective dosemay be from about 1 μg to 5 mg per dose, more preferably from about 10μg to 2 mg per dose, more preferably from about 50 μg to about 1 mg perdose, and more preferably from 100 μg to about 500 μg per dose.Preferably the therapeutically effective dose is a single dose. Alsopreferably administration of the vaccine to the mammal produces no sideeffects such as, for example, no fever or inflammation at the site ofadministration.

Another embodiment of the invention comprises methods for manufacture ofa vaccine that treats or prevents an infection caused by one or more ofB. pertussis, B. parapertussis or a serotype or strain of B. pertussisor B. parapertussis comprising combining a detoxified pertussis toxinprotein, an enzyme treated LPS, and a detoxified adenylate cyclaseprotein. Preferably the composition comprises a pharmaceuticallyacceptable carrier and/or an adjuvant.

Other embodiments and advantages of the invention are set forth in partin the description, which follows, and in part, may be obvious from thisdescription, or may be learned from the practice of the invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of the detoxification of pertussis.

DESCRIPTION OF THE INVENTION

Current acellular pertussis preparations are difficult to make and donot consistently elicit an immune response strong enough to protectvaccinated subjects. This is believed to be due to the removal of innatedanger signals present in the whole cell vaccine, but absent in thehighly purified acellular preparation. On the flip side, whole cellpertussis vaccine is reactogenic and therefore not preferred forpediatric use. A large part of the reactogenicity and toxicity residuein the pertussis toxins encoded by Pertussis Toxin (PT) and cyclase aswell as by the TLR4 agonist LPS. Both cyclase and LPS can be made intoadjuvants that are safe by disrupting the adenylate cyclase activity ofcyclase and by removing phosphate and sugars from the LPS yielding anMPL like molecule.

It has been surprisingly discovered that by engineering a pertussisstrain currently utilized as a whole-cell vaccine, the undesirableeffects from the quality immune effects a whole cell vaccine can bedecoupled. Decoupling is performed by detoxifying three componentsnamely pertussis toxin (PT), pertussis lipopolysaccharide (P-LPS), andpertussis adenylate cyclase toxin (P-ACT).

Accordingly, one embodiment of the invention is directed to a vaccinefor the treatment or prevention of infection by B. pertussis comprisingdetoxified pertussis toxin (PT), detoxified pertussis lipopolysaccharide(P-LPS), and detoxified pertussis adenylate cyclase toxin (P-ACT) PT isreleased from B. pertussis in an inactive form and binds to a cellmembrane receptor. From the receptor, PT is adsorbed into endosomes andundergoes a retrograde transport which activates the A subunit, thatthan catalyzes ADP-ribosylation of the α_(i) subunits of theheterotrimeric G protein. G protein is than prevented from interactingwith its membrane receptor and thereby prevent intracellularcommunication. The Gi subunits remain locked in an inactive state unableto inhibit adenylyl cyclase activity. This results in an increasedcellular concentration of cAMP that thus prevents biological signaling,causing a release of insulin and hypoglycemia. PT is therefore apreferred component of acellular vaccines.

Another embodiment of the invention is directed to methods of treatingor preventing an infection comprising administering a therapeuticallyeffective amount of the vaccine of the invention to a mammal. Vaccinesof the invention may be administered to patients in need in a variety ofroutes such as, for example, via injection (e.g., intramuscular,intravenous), orally by tablets, pills or liquids, nasally by spray oranother route known and available to those skilled in the art. Vaccinesof the invention may also include a pharmaceutically acceptable carriersuch as, for example, water, glycerol, alcohol, propylene glycol, fattyalcohols, triglycerides, fatty acid esters, mineral oils, liquidpetrolatum, isopropylpalmitate, polyethylene ethanol, polyoxyethylenemonolauriater, sodium lauryl sulfate, an anti-oxidant, a humectant, aviscosity stabilizer or modifier, a colorant or a flavoring agent.Preferably the vaccine of the invention is non-toxic at theconcentration utilized, and contains no adhesins, such as B. pertussisadhesins.

Detoxifying PT

PT dissociates into two parts in the endoplasmic reticulum (ER): theenzymatically active A subunit (S1) and the cell-binding B subunit. Thetwo subunits are separated by proteolytic cleavage. The B subunit willundergo ubiquitin-dependent degradation by the 26S proteasome. However,the A subunit lacks lysine residues, which are essential forubiquitin-dependent degradation. Therefore, PT subunit A will not bemetabolized like most other proteins. PT is heat-stable andprotease-resistant, but once the A and B are separated, these propertieschange. The B subunit will stay heat-stable at temperatures up to about60° C., but it is susceptible to protein degradation. PT subunit A, onthe other hand, is less susceptible to ubiquitin-dependent degradation,but is unstable at temperature of about 37° C. and above. Thisfacilitates unfolding of the protein in the ER and tricks the cell intotransporting the A subunit to the cytosol, where normally unfoldedproteins will be marked for degradation. So, the unfolded conformationwill stimulate the ERAD-mediated translocation of PT A into the cytosol.Once in the cytosol, it can bind to NAD and form a stable, foldedprotein again. Being thermally unstable is also the Achilles heel of PTsubunit A. As always, there is equilibrium between the folded andunfolded states. When the protein is unfolded, it is susceptible todegradation by the 20S proteasome, which can degrade only unfoldedproteins.

There are a number of mutations that result in detoxified PT while stillallowing the production of antibodies responsive to native PT. Pertussistoxin has five subunits, S1-S5, and most proposed mutations focus onS1-S3. Mutations in the catalytic subunit Si disable theADP-ribosylation activity of PT while leaving the structure intact andinclude Arg⁹→Lys⁹ and Glu¹²⁹→Gly¹²⁹ (see e.g., U.S. Pat. No. 7,169,399,which is incorporated by reference). The remaining four subunits of PTconstitute the B-oligomer responsible for binding cells prior to theirinvasion. Proposed mutations or deletions to the S2 and/or S3 subunitsinclude some combination of Asn¹⁰⁵, Tyr¹⁰², or Tyr¹⁰²⁻¹⁰³ of S2 and/orLys¹⁰, Tyr⁹², Lys⁹³, Lys⁹³, or Tyr¹⁰²⁻¹⁰³ of S3. These mutations hinderthe ability of PT to bind cells while still enabling the generation ofanti-PT antibodies.

Chromosomal integration of mutations involves two major steps—plasmidtransfer via bacterial conjugation from transformed E. coli followed bychromosomal integration via homologous recombination. There are a numberof recombinant strategies for accomplishing these steps, all well-knownand commercially available. A preferred method involves conjugativetransfer using an E. coli strain (e.g. SM10 as the conduit into B.pertussis) and the Life Technologies TALEN system for homologousrecombination integration into the chromosomal DNA.

Modifying P-LPS to Remove Reactogenicity

To detoxify LPS, phosphatase (e.g. alkaline phosphatase, Antarcticphosphatase, lambda protein phosphatase) is used to dephosphorylate thecore-oligosaccharide of LPS, which results in an effective means ofdetoxification of the compound. Upon induced lysis of B. pertussis,samples are treated with these phosphatases before performingcytotoxicity assays to identify preferred enzymes. Plasmids formed willtrigger enzyme expression and apoptosis upon induction. The designergenes are integrated into the B. pertussis chromosomal DNA as describedabove. This allow a production process whereby cells are cultured tosaturation, the phosphatase is induced, and upon lysis generates a mono-and aphosphoryl-LPS/Lipid A that retains adjuvant activity without toxiceffects. A similar strategy is applied using a deacylases that removeone or more acyl chains from the Lipid A core thereby reducing orremoving innate stimulatory capacity.

Replacing P-ACT with a Detoxified Version

As with PT, changes in P-ACT can be modified to generate a non-toxicversion of this protein. The dipeptide leu-gln is inserted between aminoacids 188 and 189 of the native P-ACT, which detoxifies P-ACT.Alternatively, amino acid residues 1188 to 1281 of the CyaA protein ofB. pertussis may be mutated to create a vector wherein eithermodification is used in the same manner as described in herein; to knockout native P-ACT to be replaced with a non-toxic version that maintainsadjuvanticity as well as the antigen's immunogenicity.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All references cited herein,including all publications, U.S. and foreign patents and patentapplications, are specifically and entirely incorporated by reference.The term comprising, where ever used, is intended to include the termsconsisting and consisting essentially of. Furthermore, the termscomprising, including, and containing are not intended to be limiting.It is intended that the specification and examples be consideredexemplary only with the true scope and spirit of the invention indicatedby the following claims.

1. A vaccine for the treatment or prevention of an infection in a mammalcomprising: a detoxified pertussis toxin protein; an enzyme treated LPS;and a detoxified adenylate cyclase protein.
 2. The vaccine of claim 1,wherein the infection is caused by one or more of B. pertussis, B.parapertussis or a serotype or strain of B. pertussis or B.parapertussis.
 3. The vaccine of claim 1, which is protective againstand/or treats an infection caused by one or more of B. pertussis, B.parapertussis or a serotype or strain of B. pertussis or B.parapertussis.
 4. The vaccine of claim 1, which is protective againstand/or treats an infection caused by multiple serotypes and/or strainsof B. pertussis and/or B. parapertussis
 5. The vaccine of claim 1,wherein the detoxified pertussis protein contains a mutation in one ormore of subunits S1-S6 of B. pertussis.
 6. The vaccine of claim 5,wherein the mutation comprises a substitution in S1 of glutamic acid forglycine at amino acid position 129 and a substitution of arginine forlysine at amino acid position
 9. 7. The vaccine of claim 1, whereinenzyme treated LPS comprises LPS treated with one or more alkalinephosphatases or one or more deacylases.
 8. The vaccine of claim 7,wherein the one or more phosphorylaese comprise Antarctic phosphatase orlambda protein phosphatase.
 9. The vaccine of claim 1, wherein thedetoxified adenylate cyclase protein comprised a dipeptide insertedbetween amino acids 188 and 189 of native P-ACT.
 10. The vaccine ofclaim 1, further comprising a pharmaceutically acceptable carrier. 11.The vaccine of claim 10, wherein the pharmaceutically acceptable carriercomprises water, glycerol, alcohol, propylene glycol, fatty alcohols,triglycerides, fatty acid esters, mineral oils, liquid petrolatum,isopropylpalmitate, polyethylene ethanol, polyoxyethylene monolauriater,sodium lauryl sulfate, an anti-oxidant, a humectant, a viscositystabilizer or modifier, a colorant or a flavoring agent.
 12. The vaccineof claim 1, which produces no side effects upon administration to anindividual.
 13. The vaccine of claim 12, wherein the no side effectscomprises the absence of a fever or an inflammation at the site ofadministration.
 14. A method of treating or preventing an infectioncaused by one or more of B. pertussis, B. parapertussis or a serotype orstrain of B. pertussis or B. parapertussis, in a mammal comprisingadministering to the mammal a therapeutically effective dose of avaccine, wherein the vaccine comprises a detoxified pertussis toxinprotein; an enzyme treated LPS; and a detoxified adenylate cyclaseprotein.
 15. The method of claim 14, wherein administering is viainjection, intramuscular injection, intravenous injection, oraladministration, nasal administration, or a combination thereof.
 16. Themethod of claim 14, wherein the vaccine is a liquid, a tablet, a pill ora spray.
 17. The method of claim 14, wherein the therapeuticallyeffective dose is an amount of vaccine which produces an immune responsein the mammal that is protective against and/or treats the infection.18. The method of claim 17, wherein the amount of vaccine is from about1 μg to 1 mg per dose.
 19. The method of claim 14, wherein thetherapeutically effective dose is a single dose.
 20. The method of claim14, wherein administration of the vaccine to the mammal produces no sideeffects.
 21. The method of claim 20, wherein the no side effectscomprise the absence of a fever or an inflammation at the site ofadministration.
 22. A method for manufacture of a vaccine that treats orprevents an infection caused by one or more of B. pertussis, B.parapertussis or a serotype or strain of B. pertussis or B.parapertussis comprising combining a detoxified pertussis toxin protein,an enzyme treated LPS, and a detoxified adenylate cyclase protein. 23.The method of claim 22, wherein the composition comprises apharmaceutically acceptable carrier and/or an adjuvant.